Identification of SNPs in TG and EDG1 genes and their relationships with carcass traits in Korean cattle (Hanwoo).

Vol. 39, No. 3, pp. 349-355, September 2012

CNU Journal of Agricultural Science

DOI: http://dx.doi.org/10.7744/cnujas.2012.39.3.349

Identification of SNPs in TG and EDG1 genes and
their relationships with carcass traits in Korean cattle (Hanwoo)
Muhammad Cahyadi1,2, Dyah Maharani1,3, Seung Heui Ryoo4, Seung Hwan Lee5, Jun Heon Lee1*
1

Department of Animal Science and Biotechnology, College of Agriculture and Life Sciences, Chungnam National University, Daejeon 305-764, Korea
Department of Animal Science, Faculty of Agriculture, Sebelas Maret University, Surakarta 57126, Indonesia
3
Faculty of Animal Science, Gadjah Mada University, Yogyakarta 55281, Indonesia
4
Livestock Research Institute, Government of Chungcheongnam-Do, Cheongyang 345-811, Korea
5
Hanwoo Experiment Station, National Institute of Animal Science, RDA, Pyeongchang 232-956, Korea

2

한우에 TG와 EDG1 유전자의 단일염기다형 확인 및
도체형질과의 연관성 분
카야디1,2ㆍ디아 마하라니1,3ㆍ유승희4ㆍ이승환5ㆍ이준헌1
1

2

3

충남대학교 농업생명과학대학 동물자원생명과학과, 인도네시아 세베라스마레 대학교 축산학과, 인도네시아 가자마다 대학교 축산대학,
5
충청남도 축산기술연구소, 축산과학원 한우시험장

4

Received on 31 July 2012, revised on 11 September 2012, accepted on 12 September 2012

Abstract : Thyroglobulin (TG) gene was known to be regulated fat cell growth and differentiation and the endothelial
differentiation sphingolipid G-protein-coupled receptor 1 (EDG1) gene involves blood vessel formation and known to

be affecting carcass traits in beef cattle. The aim of this study was to identify the single nucleotide polymorphisms (SNPs)
in both TG and EDG1 genes and to analyze the association with carcass traits in Korean cattle (Hanwoo). The T354C
SNP in TG gene located at the 3’ flanking region and c.-312A>G SNP located at 3’-UTR of EDG1 gene were used
for genotyping the animals using PCR-RFLP method. Three genotypes were identified in T354C SNP in TG gene and
only two AA and AG genotypes were observed for the c.-312A>G SNP in EDG1 gene. The results indicated that T354C
SNP in TG gene was not significantly associated with carcass traits. However, the c.-312A>G SNP in EDG1 gene had
significant effects on backfat thickness (BF) and yield index (YI). These results may provide valuable information for
further candidate gene studies affecting carcass traits in Korean cattle and may use as marker assisted selection for
improving the quality of meat in Hanwoo.
Key words : TG, EDG1, Carcass traits, Hanwoo

I. Introduction

of meat (Shin et al., 2012). In Korea, high marbling score
affects consumers demands for high quality meat and

Three traits, namely carcass weight (CW), long-

also determines the cost differences of the meat.

issimus muscle area (LMA) and marbling, are considered

Previously, several studies have been reported the

as important parameters in meat quality, especially

single nucleotide polymorphisms (SNPs) in the can-

in cattle in Korea (Cheong et al., 2006; Lee et al.,

didate genes which was associated with carcass traits.

2008). Marbling is known as fat deposition in meat

This candidate gene approach is one of the effective

that significantly associated with juiciness and flavor

tools to select the animals having desirable economic

*Corresponding author: Tel: +82-42-821-5779
E-mail address: junheon@cnu.ac.kr

traits. For example, the polymorphism in MC4R gene
influenced backfat thickness and marbling score,

CNU Journal of Agricultural Science 39(3), 2012. 9

349

이준헌 / 한우에

TG와

G1 유전자의 단일염기다형 확인 및 도체형질과의 연관성 분

especially the SNP marker C1786T in Hanwoo (Seong

they were regarded as a positional functional candidate

et al., 2012). Also, the MYF5 was associated with

gene for carcass traits, especially for marbling scores.

increased meat tenderness and backfat thickness of

Therefore, the aim of this study is to identify the

beef cattle in China (Ujan et al., 2011). In addition,

polymorphisms in TG and EDG1 genes and to analyze

an SNP in GHR gene was associated the moisture and

their associations with carcass traits in Korean cattle,

intramuscular fat in semi membranous muscle in beef

Hanwoo.

samples (Reardon et al., 2010). Another report confirmed that the presence of SNPs in somatostatin

II. Materials and Methods

(SST) gene was affected marbling and fat thickness,
whereas adiponectin (ADIPOQ) gene affected both rib

1. Animals and data collection

eye muscle area and fat thickness. These genes are
Seventy three longissimus thoracis Hanwoo beef

located on BTA1 in cattle (Morsci et al., 2006).
Thyroglobulin gene (TG) produces the precursor for

samples were collected for genomic DNA extraction.

thyroid hormones which regulates metabolism and

The samples provided by the slaughter house with the

affects fat cell growth and differentiation. Also, this

help of the regional Hanwoo brand association called

gene affects homeostasis of fat deports (Gan et al.,

Tobawoo . These cattle were all castrated males and

2008). This gene has been mapped on BTA14 in the

reared under same feeding conditions including the

previously known QTL region for carcass traits,

fattening period and approximately 30 months old,

especially marbling in Hanwoo (Shin and Chung, 2007).

these animals were slaughtered. Both carcass and

Hou et al. (2010) reported that 6 novel SNPs were

meat quality data were provided by the Tobawoo. The

found at the 3 flanking region in TG gene. One of

meat parameter data including raw weight (RW),

these SNPs, T354C SNP, was strongly associated with

backfat thickness (BF), longissimus muscle (LM), carcass

marbling scores. In addition, the endothelial differ-

weight (CWT), yield index (YI), yield grade (YG), and

entiation sphingolipid G-protein-coupled receptor 1

marbling score (MS) were used in this study.

(EDG1) gene, involved in blood vessel formation, is
also known to be affecting carcass traits in beef cattle

2. The DNA extraction and genotyping

(Watanabe et al., 2010). This gene has been located
in the Quantitative Trait Locus (QTL) region and

For PCR amplification, two pairs of primers were

mapped on BTA3 using radiation hybrid (RH) panel in

designed for both thyroglobulin (TG) and EDG1 genes.

Japanese Black Cattle (Yamada et al., 2006). Previous

The primers and restrictiction enzyme information

study reported by Yamada et al. (2009) showed that a SNP

are shown in Table 1.

in the 5 untranslated region (UTR) of EDG1 gene may

volume containing 50 ng/µl DNA genome, 0.8 µl of

affect marbling score in Japanese Black Cattle. Both TG
and EDG1 genes were located in QTL region. Thus,

Polymerase chain reaction was carried out in 20 µl

each primer, 1.6 µl dNTP, 2.0 µl 10X reaction buffer,

Table 1. Primers and restriction enzyme information.
Gene

Primer

PCR product size (bp) GenBank acc. No. Restriction enzyme Primer origin

TG

F: 5’-TCCCAGAGTTAGCCTCCAAG-3’
R: 5’-GACCTCACCACCTCTCATTCA-3’

709

EU591737.1

NlaIII

Hou et al.
(2011)

EDG1

F: 5’-GTCTCAGCTGCACAGATCC-3’
R: 5’-GAAGACCTCCGGCCGCGAT-3’

378

NC_007301.5

MscI

Yamada et al.
(2008)

350 농업과학연구 제39권 제3호, 2012. 9

Jun Heon Lee / Identification of SNPs in TG and EDG1 genes and their relationships with carcass traits in Korean cattle (Hanwoo)

0.2 µl HS Taq Polymerase (GenetBio, Korea), and 12.6

µl distilated water. The PCR conditions were as

These SNPs have been confirmed with the electrophoregram results. For genotyping these SNPs, PCR-

follows: 94℃ for 10 minutes, 34 cycles of 94℃ for

restriction fragment length polymorphism (RFLP) was

seconds, the annealing temperature were 55℃ (TG) and

applied. The restriction enzyme digestion was performed

62℃ (EDG1) for 30 seconds, and 72℃ for 30 seconds,

in 20 µl reaction volumes with approximately 15 µl of

followed by a further 10 minutes final extention at 72℃.

PCR products and 2 units of each restriction enzyme.

Reaction was performed using either GeneAmp PCR

The digested products were run on 3% agarose gels.

TM

system 2700 (Applied Biosystems, USA) or C1000

Thermal Cycler (BioRad, USA). The PCR products

3. Statistical analysis

were visualized by 1.5% standard agarose gels stained
with ethidium bromide (GenetBio, Korea).

Pearson s Chi-square test was used to verify the

Each PCR fragment was purified using an Accu-

Hardy–Weinberg equilibrium status for the allele and

Prep PCR Purification Kit (Bioneer, Korea). Purified

genotype frequencies. The effects of TG and EDG1

PCR products were sequenced using the same primers

genotypes on carcass traits were tested using ANOVA

for PCR reaction in an automated 3730 XL DNA Se-

(Analysis of Variance) procedure in the SPSS version

quencer (Applied Biosystems, USA). The DNA sequence

17.0 program (SPSS, USA). The following model was

of TG and EDG1 genes were compared with reference

used for the analysis the association of the genotype

sequences using the BioEdit program ver. 7.00 (Tom

and carcass traits:

®

Hall, Ibis Therapeutics, California, USA). The sequence
alignment was performed to determine the single

Yij = µ + βi + εij

Where, Yij is traits observed in the ijth animal, µ is

nucleotide polymorphism (SNP) positions in the TG

the overall mean, βi is the i genotype effect, and εij

and EDG1 genes. The T354C SNP of TG gene was

is a random error. In order to test the pairwise differ-

located in the 3 -UTR (Fig. 1A), while the c.-312A>G

ences between the genotypes, Tukey s test was also

SNP of EDG1 was located in the 5 -UTR region (Fig. 1B).

performed.

th

Fig. 1. The gene organizations for TG (A) and EDG1 (B) genes.

CNU Journal of Agricultural Science 39(3), 2012. 9

351

이준헌 / 한우에

TG와

G1 유전자의 단일염기다형 확인 및 도체형질과의 연관성 분

III. Results

EDG1 gene, only two genotypes, AA and AG, were
observed. The AA genotype was higher genotype fre-

1. Polymorphism of TG and EDG1 in population

quency than AG genotype. The Pearson s Chi-square
test was also used to verify the Hardy–Weinberg

Initially we sequenced the PCR product of both the

equilibrium status. The X2 value of T354C SNP was

TG and EDG1 genes and identified the SNPs. The

lower than 3.84, indicating this SNP was in Hardy–

T354C SNP of TG gene was used for PCR-RFLP

Weinberg equilibrium (Table 2). However, X value of

genotyping of the animals. The 709 bp of TG gene PCR

c.-312A>G SNP cannot be tested because only one GG

product was digested into 282, 190, 112, 72, 31 and

genotype was observed.

2

22 bp fragments for the TT genotype using NlaIII
restriction enzyme. On the other hand, the CC geno-

2. The association between identified

type gave 304, 190, 112, 72, and 31 bp restriction

SNPs and carcass traits in Hanwoo

enzyme fragments (Fig. 2A). In case of EDG1 gene,

Seven carcass traits, namely raw weight (RW),

c.-312A>G SNP gave no restriction site of 378 bp PCR

backfat thickness (BF), longissimus muscle (LM),

product for GG genotype. However, for the AA geno-

carcass weight (CW), yield index (YI), yield grade

type, the c.-312A>G SNP was digested into 214 bp and

(YG), marbling score (MS), were used for association

164 bp with MscI restriction enzyme (Fig. 2B).

analysis of the SNPs in TG and EDG1 genes. The

The genotype and alelle frequencies of TG and EDG1

T354C SNP in TG gene was no association with the

genes are shown in Table 2. For the TG genes, all

carcass traits. On the other hand, the c.-312A>G SNP

three genotypes (CC, TC, and TT) were observed. The

in EDG1 gene gave significant results in backfat

TC genotype has the highest genotype frequency than

thickness (BF) (PG genotypes in EDG1 gene (B).
Table 2. Genotype and alelle frequencies of TG and EDG1 SNPs in Hanwoo population.
Gene
TG

SNP

Genotype

Genotype frequency

Alelle

Alelle frequency

Chi-square (X2) test (P-Value)

T354C

CC

0.34

C

0.61

1.095 (0.05)

TC

0.54

T

0.39

　
EDG1
　

c. -312A>G

TT

0.12

AA

0.81

A

0.90

AG

0.19

G

0.10

GG

0

352 농업과학연구 제39권 제3호, 2012. 9

-

Jun Heon Lee / Identification of SNPs in TG and EDG1 genes and their relationships with carcass traits in Korean cattle (Hanwoo)

Table 3. Association analyses between identified SNPs and carcass traits in Hanwoo.
Gene
TG
　
　

SNP

Number of
Genotype
animals

T354C
　

TT

73

　

RW (kg)

BF (mm)

656.11±35.35 10.44±2.60

LM (cm2)

CW (kg)

YI (score) YG (score) MS (score)

86.56±3.84 403.33±24.16 66.45±1.81 1.78±0.44 6.63±1.33

TC

679.93±49.03 10.18±3.28

90.40±9.48 412.38±30.91 66.91±2.74 1.53±0.51 6.93±0.99

CC

670.17±43.54 9.71±4.08

89.83±10.04 408.25±33.60 67.22±2.63 1.54±0.51 6.48±1.19

P-value

EDG1 c.-312A>G
　

73

Traits (Mean ± SD)*

0.338

0.822

0.530

0.699

0.744

0.385

0.283

AA

672.71±48.15 10.63±3.43

90.14±8.67 410.47±32.95 66.63±2.53 1.61±0.49 6.82±1.09

AG

685.79±41.26 7.79±2.99

90.07±11.03 410.71±23.36 68.40±2.58 1.36±0.50 6.37±1.11

P-value

0.352

0.006**

0.981

0.98

0.023**

0.089

0.178

*

RW: Raw Weight, BF: backfat thickness, LM: longissimus muscle area, CW: carcass weight, YI: yield index, YG: yield grade,
MS: marbling score.

(PG SNP in EDG1 gene was

Both T354C SNP of TG gene and c.-312A>G SNP of

significantly associated with backfat thickness (BF)

EDG1 gene were detected by direct sequencing of the

and AA genotype was favorable for this trait. Backfat

PCR products from a number of samples and PCR-

thickness plays important role in beef carcass pre-

RFLP was applied for the genotyping of all the samples.

servation after slaughter and determines organoleptic

Previous study reported that these SNPs were strongly

characteristics for consumer (Veneroni-Gouveia et

associated with carcass quality traits in beef cattle,

al., 2011). This SNP will be valuable as a marker to

especially for the marbling scores (Hou et al., 2011;

produce animals which have high BF. However, the

Yamada et al., 2008).

c.-312A>G SNP gave no association with marbling

The homozygous CC animals in SNP position T354C

score. In contrast, study in Japanese Black Cattle, the

was higher than TT animals for TG gene. The C allele

same SNP c.-312A>G in EDG1 have no association

frequency was higher than T allele in the Hanwoo

with BF, but it was significantly associated with

samples in this study. These results suggested that

marbling score (Yamada et al., 2009; Sukegawa et al.,

Hanwoo population may have different allele fre-

2010). Other SNP in EDG1 gene, g.1471620G>T SNP,

quency, compared with the previous study reported by

was strongly associated with high marbling score.

Hou et al. (2011). Previous study reported that TG

Previous study indicated the T allele was favorable for

gene, located on BTA14, was considered as functional

high marbling score in Japanese Black Cattle (Wata-

candidate gene for fat deposition in beef (Rincker et

nabe et al., 2010). The discrepancy between Hanwoo

al., 2006). This gene have been mapped as a candidate

and Japanese Black cattle indicated that the breed

gene which effects on lipid metabolism (Moore et al.,

defferences may affect for the variation in these

2003). Also, previous study indicated that the T354C

traits. The c.-312A>G SNP was also significantly

SNP of TG gene was significantly associated with

associated with yield index (YI), where AG animals

marbling score (Hou et al., 2011). However, in a case

were favorable for YI (pG SNP in EDG1 gene and their association
with backfat thickness (BF) and yield index (YI) in
Hanwoo. However, for the practical application, larger
scale and precise analyses are needed to confirm the
marker effects.

Acknowledgements
This work was partially supported by awards from
the AGENDA project (Grant no. PJ907008062012) and
Molecular Breeding program (PJ0081882012) of Next
Generation BioGreen21 project in the National Institute
of Animal Science, Rural Development Administration
(RDA).

References
Cheong HS, Yoon DH, Kim LH, Park BL, Choi YH, Chung
ER, Cho YM, Park EW, Cheong IC, Oh SJ, Yi SG, Park
T, Shin HD. 2006. Growth hormone-releasing hormone
(GHRH) polymorphisms associated with carcass traits of
meat in Korean cattle. BMC Genetics 7: 35-40.
Gan QF, Zhang LP, Li JY, Hou GY, Li HD, Gao X, Ren HY,
Chen JB, Xu SZ. 2008. Association analysis of thyroglobulin gene variants with carcass and meat quality traits
in beef cattle. Journal of Applied Genetics 49: 251-255.
Hou GY, Yuan ZR, Ahou HL, Zhang LP, Li JY, Gao X,
Wang DJ, Gao HJ, Xu SZ. 2011. Association of thyroglobulin gene variants with carcass and meat quality traits
in beef cattle. Molecular Biology Reports 38: 4705-4708.
Lee JJ, Choi SD, Dang CG, Kang SN, Kim NS. 2011. The
effect of carcass traits on economic values in Hanwoo.
Korean Journal for Food Science of Animal Resources 31:
603-608.
Lee SH, Cho YM, Kim BS, Kim NK, Choy YH, Kim KH,
Yoon D, Im SK, Oh SJ, Park EW. 2008. Identification of
marbling-related candidate genes in M. longissimus dorsi of
high- and low marbled Hanwoo (Korean Native Cattle)
steers. BMB Reports 41: 846-851.
Li SG, Yang YX, Rhee YJ, Jang WJ, Ha JJ, Lee SK, Song YH.
2010. Growth, behavior, and carcass traits of fattening
Hanwoo (Korean Native Cattle) steers managed in different
group sizes. Asian-Australasian Journal of Animal Science

354 농업과학연구 제39권 제3호, 2012. 9

23: 952-959.
Moore SS, Li C, Basarab J, Snelling WM, Kneeland J,
Murdoch B, Hansen C, Benkel B. 2003. Fine mapping of
quantitative trait loci and assessment of positional candidate
genes for backfat on bovine chromosome 14 in a commercial line of Bos taurus. Journal of Animal Science 81:
1919-1925.
Morsci NS, Schnabel RD, Taylor JF, 2006. Association analysis
of Adiponectin and Somatostatin polymorphisms on BTA1
with growth and carcass traits in Angus cattle. Animal
Genetics 37: 554-562.
Pannier L, Mullen AM, Hamill RM, Stapleton PC, Sweeney T.
2010. Association analysis of single nucleotide polymorphisms in DGAT1, TG and FABP4 genes and intramuscular fat in crossbred Bos taurus cattle. Meat Science
85: 515-518.
Reardon W, Mullen AM, Sweeney T, Hamill RM. 2010.
Association of polymorphisms in candidate genes with colour,
water-holding capacity, and composition traits in bovine M.
Longissimus and M. Semimembranosus. Meat Science 86:
270-275.
Rincker CB, Pyatt NA, Berger LL, Faulkner DB. 2006.
Relationship among gene STAR marbling marker, intramuscular fat deposition, and expected progeny differences
in early weaned simmental steers. Journal of Animal Science
84: 686-693.
Seong J, Suh DS, Park KD, Lee HK, Kong HS. 2012.
Identification and analysis of MC4R polymorphisms and their
association with economic traits of Korean Cattle (Hanwoo).
Molecular Biology Reports 39: 3597-3601.
Shin SC, Chung ER. 2007. Association of SNP marker in the
Thyroglobulin gene with carcass and meat quality traits in
Korean Cattle. Asian-Australasian Journal of Animal Science
20: 172-177.
Shin SC, Heo JP, Chung ER. 2012. Genetic variants of the
FABP4 gene are associated with marbling scores and meat
quality grades in Hanwoo (Korean cattle). Molecular Biology
Reports 39: 5323-5330.
Ujan JA, Zan LS, Ujan SA, Adoligbe C, Wang HB. 2011. Back
fat thickness and meat tenderness are associated with a 526
T>A mutation in the exon 1 promoter region of the MYF-5
gene in Chinese Bos taurus. Genetics and Molecular Research
10: 3070-3079.
Veneroni-Gouveia G, Meirelles SL, Grossi DA, Santiago AC,
Sonstegard TS, Yamagishi ME, Matukumalli LK, Coutinho
LL, Alencar MM, Oliveira HN, Regitano LC. 2011. Wholegenome analysis for backfat thickness in a tropically adapted,
composite cattle breed from Brazil. Animal Genetics doi:10.
1111/j.1365-2052.2011.02286.x
Yamada T, Taniguchi Y, Nishimura S, Yoshioka S, Takasuga
A, Sugimoto Y, Sasaki Y. 2006. Radiation hybrid mapping
of genes showing intramuscular fat deposition-associated
expression changes in bovine musculus longissimus muscle.
Animal Genetics 37: 184-185.

Jun Heon Lee / Identification of SNPs in TG and EDG1 genes and their relationships with carcass traits in Korean cattle (Hanwoo)

Yamada T, Itoh M, Nishimura S, Taniguchi Y, Miyake T,
Sasaki S, Yoshioka S, Fujita T, Shiga K, Morita M, Sasaki
Y. 2009. Association of single nucleotide polymorphisms in
the Endothelial differentiation sphingolipid g-protein-coupled
receptor 1 gene with marbling in japanese black beef cattle.
Animal Genetics 40: 209-216.

Watanabe N, Yamada T, Yoshioka S, Itoh M, Satoh Y, Furuta
M, Komatsu S, Sumio Y, Fujita T, Sasaki Y. 2010. The T
allele at the g.1471620 G>T in the EDG1 gene associated
with high marbling in Japanese Black Cattle is at a low
frequency in breeds not selected for marbling. Animal Science
Journal 81: 142-144.

CNU Journal of Agricultural Science 39(3), 2012. 9

355